Andrzej B. Legocki

Purification and some properties of elongation factor 1 from wheat germ

Abstract The elongation factor 1 (EF1) was extensively purified from wheat germ. The purification procedure consisted of (NH 4 ) 2 SO 4 precipitation, gel filtration on Sephadex G-150, double chromatography on hydroxylapatite and finally Sephadex G-150 gel filtration. A 70-fold purification of the factor was achieved with a recovery of 30 %. The purified preparation contains three active forms of the factor differing markedly in molecular size. Determination of their molecular weights using gel filtration and gel electrophoresis techniques resulted in values of 250 000, 180 000 and 60 000. The larger EF1 forms dissociate to the single species of mol. wt 60 000, during electrophoresis in the presence of sodium dodecyl sulphate. The forms observed may represent a monomer, trimer and tetramer of a single protein unit. The most stable form of wheat germ EF1 seems to be a trimer. Some properties of the factor have been tested. Optimum pH and temperature in phenylalanyl-tRNA binding assay are 8.1 and 30 °C, respectively. Heat inactivation studies of EF1 revealed that there is a direct interaction between GTP, aminoacyl-tRNA and the factor in the absence of ribosomes. The EF1-dependent binding of phenylalanyl-tRNA to ribosomes occurs in the presence of both guanosine triphosphate and 5′-guanylyl methylene diphosphonate.

Expression of lupin nodulin genes during root nodule development

Abstract Seven lupin cDNA clones were used to study the expression of corresponding genes during nodule development by Northern blots analysis. They include six nodulin cDNAs: pLLb (lupin leghemoglobin), pLN 13, pLN 21–27, pLN 281, pLN 50, pLNGS (nodule form of glutamine synthetase GSn and root form of GS: pGS. The appearance of nodulin mRNAs during lupin nodule development showed that the nodulin sequences analysed represent a group of plant genes involved in the nitrogen fixation process rather than formation of nodule. This is based on the observation that they are activated at the time when the nodule has already been formed, prior to the onset of nitrogenase activity. The products of Lb, nodulin 21–67, the nodulin coded by pLN13 and the nodulin 281 genes appeared between 11 and 13 days after infection, whereas the nodulion coded by pLN50 and the nodule form of GS appeared 18 days after inoculation. Twenty-one days post-infection a dramatic increase in the transciption rates of all nodulin genes is observed. This phenomenon may be related to the onset of nitrogenase activity. The possible mechanism of two-step activation of nodulin genes is discussed.

Characterization of cDNA clone coding for nodulin-45 from yellow lupin (lupinus luteus)

Abstract We describe in this paper a characterization of full-length (1396 bp) nodule-specific cDNA clone (pLN-13) from yellow lupin root nodules. The mRNA corresponding to the cDNA insert appears prior to the onset of nitrogenase activity, i.e. 11 days after infection. Hybrid-released translation analysis revealed that the selected mRNA encodes a single polypeptide of molecular weight 45 000. According to the nomenclature proposed for nodulins, this protein was named nodulin-45. The primary sequence and deduced amino acid composition indicates that nodulin-45 is a highly hydrophilic protein with potential N-terminal signal peptide and two putative asparagine-linked glycosylation sites.

Acidic proteins from wheat germ ribosomes

Certain acidic proteins from both prokaryotic and eukaryotic ribosomes are involved in various steps of polypeptide synthesis. In prokaryotes, a number of studies have shown that protein S 1 directly participates in translation of synthetic and natural mRNAs at the levels of initiation [l-4] and elongation IS]. Other than Sl , acidic proteins from bacterial ribosomes whose function has aiready been determined ire proteins L7 and I_1 2 that are involved in polypeptide chain elongation (reviewed [6j). The functions of acidic proteins from eukaryotic ribosomes have so far been much less investigated than their counterparts from prokaryotes. IIt is now believed, however, that at least so&e eukaryotic acidic proteins are functionally related to their prokaryotic homologs. l’hs, functional and imhunochemical crossreactivity has been demonstrated between E. coli proteins L7 and El2 and two acidic ribosomal proteins from l-at liver [ 7 3, yeast [S], brine shrimp Artewi~ saliva [9,1Oj, Krebs HI ascite cells [I 11 as well as from clricketl liver [ 121. T!lese Gildings suggest that despite considerabble structural differences bel.ween prokaryotic s:.nd eukaryotic ribosomes, some of their acidic proteins are highly conservative functionally. Here we present a preliminary charxtcrizzticrr of acidic proteins, from wheat germ ribosomes, v/hi& so far have not been described.

Functional Characterization of Some Ribosomal Proteins from Wheat Germ

Detailed study of the topography and properties of ribosomal proteins may greatly facilitate elucidation of the function of ribosomes during the translational process. Although the basic mode of action and the structural architecture of eukaryotic ribosomes is similar to that of bacterial ribosomes, they are of higher complexity and differ significantly in a number of their structural components.